The desire to hasten the identification of potentially important drugs, catalysts, chemical and biological sensors, medical diagnostics, and other materials is a constant challenge that has prompted the use of combinatorial synthetic and screening strategies for synthesizing these materials and screening them for desirable properties. Combinatorial synthesis involves assembling a “library”, i.e. a very large number of chemically related compounds and mixtures, usually in the form of an array on a substrate surface. High throughput screening of an array involves identifying which members of the array, if any, have the desirable property or properties. The array form facilitates the identification of a particular material on the substrate. Combinatorial arrays and high-throughput screening techniques have been used to solve a variety of problems related to the development of biological materials such as proteins and DNA because the screening techniques can be used to rapidly assay many biological materials.
The binding properties of a protein largely depend on the exposed surface amino acid residues of its polypeptide chain (see, for example, Bruce Alberts et al., “Molecular Biology of the Cell”, 2nd edition, Garland Publishing, Inc., New York, 1989; and H. Lodish et al., “Molecular Cell Biology”, 4th edition, W. H. Freeman and Company, 2000). These amino acid residues can form weak noncovalent bonds with ions and molecules. Effective binding generally requires the formation of many weak bonds at a “binding site,” which is usually a cavity in the protein formed by a specific arrangement of amino acids. There should be a precise fit with the binding site for effective binding to occur.
The chemical properties and in particular, the binding properties of a protein depend almost entirely on the exposed surface amino acid residues of the polypeptide chain. These residues can form weak noncovalent bonds with other molecules. An effective binding between the protein, one example of a group of materials herein referred to as “receptors”, and the material that binds to the receptor, referred to herein as “chemical”, generally requires that many weak bonds form between the protein receptor and the chemical. Chemicals include organic molecules, inorganic molecules, salts, metal ions, and the like. The bonds between the protein and the chemical form at the “binding site” of the protein. The binding site is usually a cavity in the protein that is formed by a specific arrangement of amino acids that often belong to widely separated regions of the polypeptide chain and represent only a minor fraction of the total number of amino acids present in the chain. Chemicals should fit precisely into the binding site for effective binding to occur. The shape of these binding sites can differ greatly among different proteins, and even among different conformations of the same protein. Even slightly different conformations of the same protein may differ greatly in their binding abilities. For further discussion of the structure and function of proteins, see: Bruce Alberts et al., “Molecular Biology of the Cell”, 2nd edition, Garland Publishing, Inc., New York, 1989; and H. Lodish et al., “Molecular Cell Biology”, 4th edition, W. H. Freeman and Company, 2000.
After a receptor array is prepared, it is screened to determine which members have the desirable property or properties. U.S. Pat. No. 5,143,854 to M. C. Pirrung et al. entitled “Large Scale Photolithographic Solid Phase Synthesis of Polypeptides and Receptor Binding Screening Thereof”, which issued Sep. 1, 1992, hereby incorporated by reference, describes one such screening method. A polypeptide array is exposed to a ligand (an example of a chemical) to determine which members of the array bind to the ligand. The ligands described are radioactive, or are “tagged”, i.e. attached via one or more chemical bonds to a chemical portion that fluoresces when exposed to non-ionizing, ultraviolet radiation. Thus, the attached portion, i.e. the tag, makes the chemical visible by interrogation with ultraviolet radiation. Tagged molecules have also been used to aid in sequencing immobilized polypeptides as described, for example, in U.S. Pat. No. 5,902,723 to W. J. Dower et al. entitled “Analysis of Surface Immobilized Polymers Utilizing Microfluorescence Detection,” which issued May 11, 1999. Immobilized polypeptides are exposed to molecules labeled with fluorescent tags. The tagged molecules bind to the terminal monomer of a polypeptide, which is then cleaved and its identity determined. The process is repeated to determine the complete sequence of the polypeptide.
It is generally assumed that the attachment of a fluorescent tag to a chemical only serves to make visible the otherwise invisible chemical, and does not alter its binding properties. Since it is well known that even small changes to the structure of a molecule could affect its function, this assumption that a tagged chemical, i.e. a “surrogate”, has the same binding affinity as the untagged chemical may not be a valid one. Small structural changes that accompany even a conformational change of a receptor have been known to affect the binding affinity of the receptor. The tagged surrogates are structurally different from their untagged counterparts, and these structural differences could affect their binding affinities. Since binding affinities derived using tagged surrogates are suspect, the binding properties of receptors and chemicals should be evaluated using the untagged chemical or receptor and not with a tagged surrogate.
Pharmaceutical chemicals are the active ingredients in drugs, and it is believed that their therapeutic properties are linked to their ability to bind to one or more binding sites. The shapes of these binding sites may differ greatly among different proteins, and even among different conformations of the same protein. Even slightly different conformations of the same protein may differ greatly in their binding abilities. For these reasons, it is extremely difficult to predict which chemicals will bind effectively to proteins.
Development and manufacturing for a new pharmaceutical chemical for a drug, i.e. drug development, generally involves determining the binding affinities between a potential pharmaceutical chemical (preferably a water soluble organic chemical that can dissolve into the blood stream) and a receptor (generally a biological material such as an enzyme or non-enzyme protein, DNA, RNA, human cell, plant cell, animal cell, and the like) at many stages of the drug development process. The receptor may also be a microorganism (e.g. prion, virus, bacterium, spores, and the like) in whole or in part. The drug development process typically involves procedures for combining potential pharmaceutical chemicals with receptors, detecting chemical binding between the potential pharmaceutical chemicals and the receptors and determining the binding affinity and kinetics of binding of a receptor to a chemical to form a complex or the kinetics of release of a bound chemical from a complex. The binding affinity is defined herein as the associative equilibrium constant Ka for the following equilibrium expression:m (chemical)+receptorchemical-receptor complex
The binding affinity, Ka, is defined by equation (1) below.Ka=[chemical-receptor complex]/[receptor][chemical]m  (1)In equation (1), [chemical-receptor complex] is the concentration in moles per liter of the chemical-receptor complex, [receptor] is the concentration in moles per liter of the receptor, ‘m’ is the number of molecules of chemical that bind to each molecule of receptor, and [chemical] is the concentration in moles per liter of the chemical. Any effects due to concentration can be simplified if the concentration of chemical used were the same for all receptors.
Nowadays, the drug development process may involve the rapid screening of hundreds or thousands of chemicals in order to identify a “lead compound,” which is one of the many tested that binds very strongly, i.e. has a high binding affinity, with a particular receptor. After such a lead compound has been identified, then other potential pharmaceutical chemicals similar in structure to the lead compound, which are referred to herein as “analogs” of the lead compound, are synthesized and tested in order to determine which of these chemicals, if any, exhibits an even higher binding affinity.
The preparation and high-throughput screening of biological arrays is exemplified by the following papers: H. Zhu and M. Snyder, “Protein Chip Technology (Review),” Current Opinion in Chemical Biology, vol. 7, pp. 55-63, (2003); G. MacBeath and S. L. Schreiber, “Printing Proteins As Microarrays For High-Throughput Function Determination,” Science, vol. 289, pp. 1760-1763, (2000); E. T. Fung et al., “Protein biochips for differential profiling,” Analytical Biotechnology, Vol. 12, pp. 65-69, (2001); T. Kukar et al., “Protein Microarrays to Detect Protein-Protein Interactions Using Red and Green Fluorescent Proteins,” Analytical Biochemistry, vol. 306, pp. 50-54, (2002); G. Y. J. Chen et al., “Developing a Strategy for Activity-Based Detection of Enzymes in a Protein Microarray,” ChemBioChem, pp. 336-339, (2003); K. Martin et al., “Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye,” Proteomics, Vol. 3, pp. 1244-1255, (2003); J. Burbaum and G. M. Tobal, “Proteomics in drug discovery,” Current Opinion in Chemical Biology, Vol. 6, pp. 427433, (2002); I. A. Hemmilä and P. Hurskainen, “Novel detection strategies for drug discovery,” Drug Discovery Today, Vol. 7, pp. S150-S156, (2002), Pages S150-S156; all incorporated herein by reference.
Some screening methods are described in the following patents, all of which are hereby incorporated by reference.
U.S. Pat. No. 6,147,344 to D. Allen Annis et al. entitled “Method for Identifying Compounds in a Chemical Mixture”, which issued Nov. 14, 2000, describes a method for automatically analyzing mass spectrographic data from mixtures of chemical compounds.
U.S. Pat. No. 6,344,334 to Jonathan A. Ellman et al. entitled “Pharmacophore Recombination for the Identification of Small Molecule Drug Lead Compounds,” which issued Feb. 5, 2002, describes a method for identifying a drug lead compound by contacting target biological molecules with cross-linked binding fragments.
U.S. Pat. No. 6,395,169 to Ole Hindsgaul et al. entitled “Apparatus for Screening Compound Libraries,” which issued May 28, 2002, describes an apparatus that employs frontal chromatography combined with mass spectrometry to identify and rank members of a library that bind to a target receptor.
Other current high-throughput screening methods, along with their associated drawbacks, are listed in TABLE 1 below, which is taken from the following paper: H. Zhu and M. Snyder, “Protein Chip Technology (Review),” Current Opinion in Chemical Biology, vol. 7, pp. 55-63, (2003).
TABLE 1Detection TechniqueProbe LabelingData AcquirementELISA (Enzyme-LinkedEnzyme-Linked AntibodiesCCD ImagingImmunosorbent Assay)Isotopic LabelingRadioisotope Labeled AnalyteX-Ray Film Or PhospoimagerSandwich ImmunoassayFluorescently LabeledLaser ScanningAntibodiesSurface Plasmon ResonanceReceptor must be attached toRefractive Index Changea surfaceNon-Contact Atomic ForceNoneSurface Topological ChangeMicroscopyPlanar WaveguideFluorescently LabeledCCD ImagingAntibodiesSELDI (Surface EnhancedNoneMass SpectrometryLaser Desorption IonizationMass Spectrometry)ElectrochemicalMetal-Coupled AnalyteConductivity Measurement
TABLE 1 shows that most of the listed screening methods have the same drawback: they require either radiolabeled chemicals, chemicals that have been altered with a fluorescent tag, or chemicals that have been altered with a metal tag.
X-ray fluorescence (XRF) spectrometry is a powerful spectroscopic technique that has been used to determine the elements that are present in a chemical sample, and to determine the quantity of those elements in the sample. The underlying physical principle of the method is that when an atom of a particular element is irradiated with X-ray radiation, the atom ejects a core electron such as a K shell electron. The resulting atom is in then an excited state, and it can return to the ground state by replacing the ejected electron with an electron from a higher energy orbital. This is accompanied by the emission of a photon, i.e. X-ray fluorescence, and the photon energy is equal to the difference in the energies of the two electrons. Each element has a characteristic set of orbital energies and therefore, a characteristic X-ray fluorescence (XRF) spectrum.
An XRF spectrometer is an apparatus capable of irradiating a sample with an X-ray beam, detecting the X-ray fluorescence from an element included in the sample, and using the X-ray fluorescence to determine which elements are present in the sample and providing the quantity of these elements. A typical, commercially available X-ray fluorescence spectrometer is the EDAX Eagle XPL energy dispersive X-ray fluorescence spectrometer, equipped with a microfocus X-ray tube, lithium drifted silicon solid-state detector, processing electronics, and vendor supplied operating software. In principle, any element may be detected and quantified with XRF.
U.S. patent application 20030027129 entitled “Method for Detecting Binding Events Using Micro-X-ray Fluorescence Spectrometry,” incorporated by reference herein, describes a method for detecting binding events using micro-x-ray fluorescence spectrometry. According to the '129 patent application, receptors are exposed to at least one potential chemical and arrayed on a substrate support. Each member of the array is exposed to X-ray radiation. The magnitude of a detectable x-ray fluorescence signal for at least one element can be used to determine whether a binding event between a chemical and a receptor has occurred, and can provide information related to the extent of binding between the chemical and receptor.
U.S. patent application 20040017884 entitled “Flow Method And Apparatus For Screening Chemicals Using Micro X-Ray Fluorescence,” which was published on Jan. 29, 2004, incorporated by reference herein, describes a method for identifying binding events between potential pharmaceutical chemicals and receptors (e.g. proteins). The method involves modifying a mixture of potential pharmaceutical chemicals by adding at least one receptor to the mixture. After allowing sufficient time for any bound complex between any of the potential pharmaceutical chemicals and any of the receptors to form, if such a complex can form, the resulting solution is flow separated into at least two components. Each component is exposed to an x-ray excitation beam. If the exposed component emits a detectable x-ray fluorescence signal, that component is isolated. The identity of any isolated component can be determined using one or more standard analytical techniques, such as gas chromatography, liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, infrared spectroscopy, ultraviolet spectroscopy, visible spectroscopy, elemental analysis, cell culturing, immunoassaying, and the like.
Effective drugs are selective drugs; they bind to a specific desired receptor, bypassing other receptors, to produce a desired therapeutic effect. This way, they target a specific disease in the body with minimal side effects; selectivity provides efficacy without undesirable side effects.
The therapeutic index is a measure of drug selectivity that is ordinarily calculated from data obtained from experiments with animals. The therapeutic index of the drug is typically defined as the ratio of two numbers, LD50/ED50, where LD50 is the dose of a drug that is found to be lethal (i.e. toxic) for 50 percent of the population of animals used for testing the drug, and ED50 is the dose of the drug that is found to have a therapeutic effect for 50 percent of that population. More broadly, it is a measure of the how much of the drug is needed to produce a harmful effect relative to the amount needed to produce a beneficial effect. The ratio LD50/ED50 is therefore, a measure of the approximate “safety factor” for a drug; a drug with a high therapeutic index can presumably be administered with greater safety than one with a low index.
Estimating the therapeutic index of a chemical involves measuring the binding affinity of a chemical to a first receptor, and measuring the binding affinity of the same chemical to a second receptor. After measuring these binding affinities, the ratio of the binding affinity of the chemical divided by the amount of first receptor versus the binding affinity of the chemical divided by the amount of the second receptor is determined. This ratio is provides an estimate of the “therapeutic index”. For an example of using DNA arrays with optical fluorescence high-throughput screening to estimate a therapeutic index, see for example, R. Ulrich and S. H. Friend, “Toxicogenomics and Drug Discovery: Will New Technologies Help Us Produce Better Drugs?,” Nature Reviews Drug Discovery, v.1, pp.84-88 (2002), incorporated herein by reference.
There remains a need for simpler methods for measuring binding affinities and selectivities, estimating the therapeutic index of a chemical, and for expediting drug manufacturing,
Therefore, an object of the present invention is to provide a method for measuring binding affinities of chemicals.
Another object of the present invention is to provide a method for measuring selectivity of chemicals binding to receptors.
Yet another object of the invention is to provide a method for estimating the therapeutic index of a chemical.
Still another object of the invention is to provide a method that employs x-ray fluorescence for drug manufacture.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.